Neurons communicate at chemical synapses. Synapses assemble neurons into networks of neurons and these networks create the remarkable abilities of the nervous system to perceive the world, process information, and direct behavior. Synapses also endow the nervous system with the ability to change over time - that is, to learn. Unconventional neuromodulators, such as nitric oxide, endocannabinoids, and prostaglandins, have been known about for many years;however, their participation in synaptic modulation at the vertebrate neuromuscular junction (NMJ), where a motor nerve synapses onto a muscle cell, is a more recent finding. Despite its apparent simplicity, the NMJ employs a surprisingly complex array of signaling pathways that modulate its activity. Using a muscle from the green anole lizard, which has been shown by my laboratory to undergo a biphasic modulation of neurotransmitter release that involves nitric oxide and the endocannabinoid 2-Arachidonoylglycerol (2-AG), will be used to address five questions regarding the mechanisms and/or roles of these unconventional neuromodulators. The first question will determine whether the synaptic depression mediated by 2-AG improves neuromuscular endurance by reducing ACh release during periods of intense, unremitting activity (e.g. exercise). If true, this will not only reveal a basic function of endocannabinoids at the NMJ, but may also have important implications for the therapeutic use of modulators of the endocannabinoid system (e.g. rimonabant). Nitric oxide (NO) is essential for synaptic modulation induced by the activation of muscarinic receptors at the NMJ. In fact, disruption of NO synthesis at the NMJs of humans with muscular dystrophy may contribute to fatigue. The second question will use a fluorescence-based technique for measuring NO to determine whether the muscle is the source of the NO that is essential for muscarinic-receptor dependent modulation at the NMJ. The third question follows closely from the second and will determine whether glutamate or the dipeptide N-acetylaspartylglutamate (NAAG) activates the synthesis of NO. NAAG has been implicated in numerous pathologies including pain, traumatic brain injury, schizophrenia and stroke. Although vertebrate motor neurons have long been known to express high levels of NAAG, the function of this peptide at the NMJ has not been rigorously explored. The final two questions will focus on the function of perisynaptic Schwann cells (PSCs), glial cells intimately associated with the pre- and postsynaptic elements of the NMJ. Preliminary work has implicated the prostaglandin PGE2-G in the second phase of muscarinic modulation at the NMJ. Activation of muscarinic ACh receptors is predicted to induce the synthesis of the enzyme cyclooxygenase (COX) in the PSCs, which will then convert 2-AG to PGE2-G. This prediction will be tested by measuring levels of COX mRNA using quantitative PCR and determining its location using fluorescence in situ hybridization. These studies, along with an electrophysiological investigation of NMJs at which the PSCs have been acutely ablated by a complement-mediated procedure, will further our knowledge of glial cell function in synaptic plasticity at the NMJ with likely implications for synaptic modulation in the peripheral and central nervous system. PUBLIC HEALTH RELEVANCE: Using electrophysiological, optical and molecular genetic approaches at the vertebrate neuromuscular junction this research will explore five fundamental hypotheses about the involvement of unconventional synaptic plasticity at the chemical synapse. The findings from this research will have direct relevance to the therapeutic use of modulators of the endocannabinoid system (e.g. rimonabant). It will also provide basic knowledge that may better inform our understanding of muscular dystrophy and numerous other diseases that are caused by the dysfunction of glial cells or the peptide N-acetylaspartylglutamate (NAAG).